The determination of molecular weight is essential for preliminary assurance that the recombinant product is synthesized as expected. Molecular weight measurement can indicate potential sequence variation and provide a high level assessment of post-translational modification, including glycosylation. Our high-resolution LC-MS methods provide accurate molecular weight information and complements our size exclusion chromatographic methods.
Intact Mass Analysis and mAb Subunit Analysis by LC/MS
We have methods to assess the mass of the intact protein, deglycosylated protein and reduced protein.
Intact mass analysis is the process of determining the molecular weight of a protein. Comparison of the observed mass to the mass expected from the primary sequence provides a preliminary assurance of amino acid composition. Intact mass analysis also provides information on the nature of the main protein glycoforms, as well as other major modifications.
Deglycosylation of an IgG prior to analysis by LC/MS removes the heterogeneity associated with
N-glycosylation. This is employed to assess the mass of the protein backbone without the glycans attached. Together with the intact mass data, this method provides indirect validation of the primary structure.
LC/MS analysis of the reduced monoclonal antibody is used to confirm the molecular weight of the individual light and heavy chains. In addition to confirming the data for the intact and deglycosylated mass, the analysis of smaller protein fragments means that the method is more sensitive to less abundant modifications.
A comparative approach can also be implemented for developers of biosimilar mAb therapeutics. We can run side by side analysis of the biosimilar material to a reference material. This can highlight examples whereby issues may arise in proving fingerprint-like similarity. Learn more about how structure affects functional activity of biosimilar monoclonal antibodies by downloading our scientific poster.
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